Compounds and methods for treating pain

ABSTRACT

The present disclosure relates to, among other things, compounds and methods for treating neuropathic pain, ocular pain, ocular inflammation, and/or dry eye and methods of detecting mutations in specific G-protein coupled receptors, such as missense mutations, and determining the extent to which these mutations alter the pharmacological response of the G-protein coupled receptor.

RELATED APPLICATIONS

This application claims priority to, and benefit of, U.S. provisionalpatent application No. 62/253,094, filed Nov. 9, 2015, the contents ofwhich are hereby incorporated by reference in its entirety.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “ONTG-002-001WO-SequenceListing.txt”, which was created on Oct. 28, 2016 and is 1.54 KB in size,are hereby incorporated by reference in their entireties.

BACKGROUND OF THE DISCLOSURE

There is a variety of pain conditions including neuropathic pain, ocularpain, and ocular inflammation.

Neuropathic pain is a complex, chronic pain state that usually isaccompanied by tissue injury. With neuropathic pain, the nerve fibersthemselves might be damaged, dysfunctional, or injured. These damagednerve fibers send incorrect signals to other pain centers. The impact ofa nerve fiber injury includes a change in nerve function both at thesite of injury and areas around the injury. Neuropathic pain is aserious health problem that affects millions of people worldwide andoccurs in as much as 7% of the general population. The management ofneuropathic pain in patients is complex with an estimated 40-60% ofindividuals refractive to existing analgesic therapies. The agingpopulation, the diabetes epidemic, and patients with cancer and AIDS allcontribute to the prevalence of intractable neuropathic pain,highlighting the pressing need to develop novel therapeutics for thiscondition.

The eye is heavily innervated by sensory nerve fibers, and inflammatory,ischemic, and even neoplastic involvement of the eye and orbit canproduce pain. Ophthalmic causes of eye pain include dry eyes and otherforms of keratitis, acute angle-closure glaucoma, and intraocularinflammation. Keratitis sicca, or dry eye, is a very common cause ofophthalmic discomfort. These conditions are most commonly diagnosedthrough examination of the cornea, anterior segment, and anteriorvitreous by slit lamp. Exacerbated by visual tasks that decrease blinkfrequency, especially work on the computer, it has various causes andresults from conditions that either decrease tear production or increasetear evaporation. Dry eye is one of the characteristic features of theautoimmune Sjögren syndrome. Evidence of fluorescein or rose bengalstaining, abnormal tear breakup time, or decreased Schirmer test mayhelp confirm dry eye syndrome. Posterior segment examination withindirect ophthalmoscopy or slit-lamp biomicroscopy may reveal evidenceof choroidal or retinal inflammation or posterior scleritis.

The present disclosure addresses the need of patients suffering fromvarious pain conditions, such as neuropathic pain, ocular pain, ocularinflammation, and/or dry eye.

SUMMARY OF THE DISCLOSURE

The present disclosure provides compositions and methods for treating orameliorating at least one symptom of neuropathic pain, ocular pain,ocular inflammation, and/or dry eye.

The present disclosure provides a composition comprising a lipidatedbovine adrenal medulla peptide 8-22 (BAM8-22) peptide analog. Thecomposition can comprise a BAM8-22 peptide, a PEG-8 linker with aLys-Gly-Gly (KGG) spacer, and a palmitic acid membrane anchor. TheBAM8-22 peptide can comprise the amino acid sequence of SEQ ID NO:1. TheBAM8-22 peptide and the KGG spacer can comprise the amino acid sequenceof SEQ ID NO:2. The PEG-8 linker and the palmitic acid membrane anchorcan be coupled to the amino side chain group of the C-terminal lysine ofthe BAM8-22 peptide.

The BAM8-22 peptide can comprise at least one amino acid modification.The at least one amino acid modification can reduce or inhibit proteaseactivity. The protease activity can be serine protease activity. The atleast one amino acid modification can be at position 15 of SEQ ID NO:1.The modification at position 15 of SEQ ID NO:1 can be a M to Asubstitution (M15A). The at least one amino acid modification can be atposition 17 of SEQ ID NO:1. The modification at position 17 of SEQ IDNO:1 can be a Y to W substitution (Y17W). The BAM8-22 peptide cancomprise at least two amino acid modifications. The at least two aminoacid modifications can be at positions 15 and 17 of SEQ ID NO:1. Themodification at position 15 of SEQ ID NO:1 can be a M to A substitution(M15A) and the modification at position 17 of SEQ ID NO:1 can be a Y toW substitution (Y17W).

The present disclosure also provides a composition comprising alipidated γ2-melanocyte stimulating hormone (γ2-MSH) peptide analog. Thecomposition can comprise a γ2-MSH peptide, a PEG-8 linker, and apalmitic acid membrane anchor. The γ2-MSH peptide can comprise the aminoacid sequence of SEQ ID NO:3. The PEG-8 linker and the palmitic acidmembrane anchor can be coupled to the N-terminus of the γ2-MSH peptide.The γ2-MSH peptide can comprise at least one amino acid modification.The at least one amino acid modification can reduce or inhibit proteaseactivity.

The present disclosure also provides pharmaceutical compositioncomprising any of the compositions of the present disclosure (e.g.,lipidated BAM8-22 peptide or lipidated γ2-MSH peptide or a combinationthereof) and a pharmaceutically acceptable carrier.

The present disclosure also provides a method of treating neuropathicpain in a subject in need thereof comprising administering atherapeutically effective amount of any of the compositions of thepresent disclosure (e.g., lipidated BAM8-22 peptide or lipidated γ2-MSHpeptide or a combination thereof).

The present disclosure also provides a method of treating ocular pain ina subject in need thereof comprising administering a therapeuticallyeffective amount of any of the compositions of the present disclosure(e.g., lipidated BAM8-22 peptide or lipidated γ2-MSH peptide or acombination thereof).

The present disclosure also provides a method of treating ocularinflammation in a subject in need thereof comprising administering atherapeutically effective amount of any of the compositions of thepresent disclosure (e.g., lipidated BAM8-22 peptide or lipidated γ2-MSHpeptide or a combination thereof).

The present disclosure also provides a method of treating dry eye in asubject in need thereof comprising administering a therapeuticallyeffective amount of any of the compositions of the present disclosure(e.g., lipidated BAM8-22 peptide or lipidated γ2-MSH peptide or acombination thereof).

In some embodiments, the peptide or peptide analog of the presentdisclosure is cyclized. For example, the BAM8-22 peptide or lipidatedBAM8-22 peptide analog is cyclized. In yet another example, the γ2-MSHpeptide or lipidated γ2-MSH peptide analog is cyclized.

Any aspect or embodiment described herein can be combined with any otheraspect or embodiment as disclosed herein. While the disclosure has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the disclosure, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic of the MrgprX1 seven transmembrane domainstructure highlighting the positions of MrgprX1 missense mutations. FIG.1B is a schematic representation of an MTL and a SMAL in relation to aGPCR.

FIG. 2 is a graph showing that the R131S MrgprX1 variant demonstratesreduced endogenous ligand mediated signaling.

FIG. 3A is a graph showing that Type I tethered BAM8-22 is active on theWT receptor. FIG. 3B is a graph showing that type II tethered γ2-MSH isactive on the WT receptor. FIG. 3C is a graph showing that lipidatedBAM8-22 exhibited increased potency compared to the correspondingsoluble peptide. FIG. 3D is a graph showing that lipidated γ2-MSHexhibited increased potency compared to the corresponding solublepeptide.

FIG. 4A is a graph showing that the R131S variant displays negligiblesignaling levels with tethered BAM8-22. FIG. 4B is a graph showing thatthe R131S variant displays reduced signaling with lipidated BAM8-22.

FIG. 5A is a graph showing that when stimulated with tethered γ2-MSH,the R131S and H133R variants exhibit decreased signaling levels comparedto the wild type receptor.

FIG. 5B is a graph showing that when stimulated with lipidated γ2-MSH,the R131S and H133R variants exhibit increased signaling levels comparedto the wild type receptor.

FIG. 6 is a graph showing that the MrgprX1 variant R131S exhibitsdecreased ligand-independent signaling.

FIG. 7A is a graph showing that both the R131S and H133R variantsexhibit levels of surface expression comparable to wild-type MrgprX1.FIG. 7B is a graph showing that both the R131S and H133R variantsexhibit levels of total expression comparable to wild-type MrgprX1.

FIG. 8 is a graph showing that recombinant membrane tethered BAM8-22activates MrgprX1.

FIG. 9 is a graph showing that the lipidated BAM8-22 analog (‘1-BAM’)has ˜100 fold higher potency than its endogenous BAM8-22 counterpart(‘s-BAM’).

FIG. 10 is a schematic showing amino acid positions in the BAM8-22peptide considered candidates for modification.

FIG. 11 is a schematic showing amino acid positions in the BAM8-22predicted to be specifically cleaved by protease family members(underlined).

FIG. 12 is a graph showing exposure of ligands to endogenous peptidasesin the presence of live cells.

FIG. 13 is a graph showing the inhibitory effect of soluble BAM8-22 onneuropathic pain in vivo.

DETAILED DESCRIPTION OF THE DISCLOSURE

In one aspect, the present disclosure provides a composition comprisinga lipidated BAM8-22 peptide analog.

In another aspect, the present disclosure provides a compositioncomprising a BAM8-22 peptide, a PEG-8 linker with a KGG spacer, and apalmitic acid membrane anchor. As used herein, the term “PEG” refers topolyethylene glycol.

In another aspect, the present disclosure provides a compositioncomprising a lipidated γ2-MSH peptide analog.

In another aspect, the present disclosure provides a compositioncomprising a γ2-MSH peptide, a PEG-8 linker, and a palmitic acidmembrane anchor.

In another aspect, the present disclosure provides a method of treatingneuropathic pain in a subject in need thereof comprising administering atherapeutically effective amount of any of the compositions of thepresent disclosure (e.g., lipidated BAM8-22 peptide or lipidated γ2-MSHpeptide or a combination thereof).

In another aspect, the present disclosure also provides a method oftreating ocular pain in a subject in need thereof comprisingadministering a therapeutically effective amount of any of thecompositions of the present disclosure (e.g., lipidated BAM8-22 peptideor lipidated γ2-MSH peptide or a combination thereof).

In another aspect, the present disclosure also provides a method oftreating ocular inflammation in a subject in need thereof comprisingadministering a therapeutically effective amount of any of thecompositions of the present disclosure (e.g., lipidated BAM8-22 peptideor lipidated γ2-MSH peptide or a combination thereof).

In another aspect, the present disclosure also provides a method oftreating dry eye in a subject in need thereof comprising administering atherapeutically effective amount of any of the compositions of thepresent disclosure (e.g., lipidated BAM8-22 peptide or lipidated γ2-MSHpeptide or a combination thereof).

In another aspect, the present disclosure also provides a method ofdetecting mutations in specific G-protein coupled receptors using any ofthe compositions of the present disclosure (e.g., lipidated BAM8-22peptide or lipidated γ2-MSH peptide or a combination thereof).

As described in further detail herein, there is an unmet need for novelstrategies to treat pain, in particular neuropathic pain, a diseasewhich affects millions of people worldwide and occurs in as much as 7%of the population. The management of patients with neuropathic pain iscomplex, with many patients not responding to treatment or onlyexperiencing partial relief. At the extreme, there is a substantialsubpopulation with moderate to severe chronic refractory pain wherethere is an urgent need for more effective, long acting therapeutics.

The transmission of pain is mediated in part by primary sensory neuronsof the dorsal root ganglia. Although the mediators of pain are complex,selected G protein-coupled receptors (GPCRs) have been implicated in themodulation of nociception. Recently, a group of GPCRs, the Mrgprs (alsodenoted Mrgpr/SNSR) were discovered in specific subsets of sensoryneurons. It has been determined that mouse MrgprC11, rat MrgprC, andhuman MrgprX1 were three orthologous receptors that are activated by thesame ligand. Stimulation of these receptors with bovine adrenal medulla8-22 peptide (BAM8-22), a gene product of the proenkephalin A gene,results in activation of Gαq leading to increases in intracellularcalcium. In mouse and rat models of neuropathic pain (chronicconstriction injury and spinal nerve ligation, respectively),intrathecal administration of BAM8-22 attenuates mechanical allodynia.Importantly, BAM8-22 administration did not affect baseline nociception,thereby not compromising protective physiological pain. One of thesubstantial limitations of using endogenous peptides as therapeutics istheir short half-life. In the above mentioned neuropathic pain models,the effect of intrathecal BAM8-22 administration was transient with ashort window of therapeutic efficacy.

The present disclosure provides long-acting, high potency, stablepeptides as modulators of GPCRs. The present disclosure providesmodified endogenous bovine adrenal medulla peptide 8-22 (BAM8-22) andmodified endogenous γ2-melanocyte stimulating hormone (γ2-MSH) togenerate higher potency analogs that anchor in the cell membrane andprovide local activity. Specifically, the compositions of the presentdisclosure include, but are not limited to, BAM8-22 and γ2-MSH lipidmodified to include a lipid-anchor. Additionally, the amino acids inthese compositions of the present disclosure can be further modified toenhance protease resistance.

The present disclosure also provides methods of producing thecompositions provided herein. One of the advantages of the methods ofthe present disclosure is starting with a recombinant system that allowsfor rapid optimization of a peptide. In Step 1, a membrane tetheredligand (MTL) is created using a peptide sequence known to activate aGPCR of interest. An alanine scan is performed to determine amino acidpositions amenable to substitution. Optimized variants are created byaltering selected amino acid positions to enable protease resistancewhile maintaining activity. Once an optimized ligand is identified, inStep 2 this peptide is incorporated as a component of a syntheticmembrane anchored ligand (SMAL) which enables delivery as a solublemolecule. The components of the SMAL (peptide-linker-anchor) can each bevaried to further fine tune pharmacological activity, as needed. Theleft side of FIG. 1B shows a MTL-GPCR interaction. An MTL cDNA constructencodes a single protein comprised of three domains: (i) a peptideligand, (ii) a flexible protein linker which includes an epitope tag(enabling detection of expression), and (iii) a transmembrane domain(TMD). The right side of FIG. 1B shows a SMAL-GPCR interaction. A SMALincludes the following domains (i) a peptide ligand, (ii) a syntheticlinker (e.g. PEG8), and (iii) a lipid anchor (e.g. palmitic acid) whichinserts into the membrane where it is locally applied (e.g. into theintrathecal space).

This approach is utilized to enhance the stability and activity of thecompositions of the present disclosure. This approach also providesadditional advantages.

The recombinant nature of MTLs enables efficient generation of candidatepeptides (without the need for synthesis and purification). Theproximity of membrane anchored ligands to cognate receptors enhances‘effective local concentration’, a major advantage in generating highaffinity ligands. The recombinant MTL system enables the generation andcharacterization of protease resistant peptides. SMAL stability can befurther enhanced through the introduction of unnatural amino acids thatare tolerated at selected positions. The cassette configuration of SMALs(peptide-linker-anchor) offers multiple opportunities (e.g. modifyingpeptide, linker length, and/or nature of membrane anchor) to fine tunecompound function (e.g. efficacy, potency, half-life, proteaseresistance). The chronic constriction injury model provides a rapidreadout of therapeutic efficacy in vivo. Combined with the modularMTL/SMAL system, this allows for a rapid identification of highly potentcompositions.

The peptide or peptide analog described herein can be cyclized. Such“cyclic peptides” have intramolecular links which connect two aminoacids. Cyclic peptides are often resistant to proteolytic degradationand are thus good candidates for oral administration. The intramolecularlinkage may encompass intermediate linkage groups or may involve directcovalent bonding between amino acid residues. In some embodiments, theN-terminal and C-terminal amino acids are linked. In other embodiments,one or more internal amino acids participate in the cyclization.Cyclization can be, for example, but not by way of limitation, via adisulfide bond between two cysteine residues or via an amide linkage.The cysteine residues can be native or artificially introduced to thepeptide. For example, cysteine residues can be introduced at theamino-terminus and/or carboxy-terminus and/or internally such that thepeptide to be cyclized. Alternatively, a cyclic peptide can be obtainedby forming an amide linkage. For example, an amide linkage can beachieved by the following protocol: (1) an allyl protected amino acid,such as aspartate, glutamate, asparagine or glutamine, can beincorporated into the peptide as the first amino acid; (2) then theremaining amino acids are coupled on to form a cycle. Other methodsknown in the art may be employed to cyclize peptides of the disclosure.For example, cyclic peptides may be formed via side-chain azide-alkyne1,3-dipolar cycloaddition (Cantel et al. J. Org. Chem., 73 (15),5663-5674, 2008, incorporated herein by reference). Cyclization ofpeptides may also be achieved, e.g., by the methods disclosed in U.S.Pat. Nos. 5,596,078; 4,033,940; 4,216,141; 4,271,068; 5,726,287;5,922,680; 5,990,273; 6,242,565; and Scott et al. PNAS. 1999. vol. 96no. 24 P. 13638-13643, which are all incorporated herein by reference.In some embodiments, the intramolecular link is a disulfide bond mimicor disulfide bond mimetic which preserves the structure that would beotherwise be created by a disulfide bond.

In one embodiment, the BAM8-22 peptide or analog thereof is cyclized. Inone embodiment, the γ2-MSH peptide or analog thereof is cyclized.

The Mas-related G protein-coupled receptor X1 (MrgprX1) is a human GPCRexpressed in dorsal root ganglia (DRG) neurons (Dong et al., Cell106:619-32, 2001; Lembo et al., Nat Neurosci 5:201-9, 2002). Theendogenous ligands bovine adrenal medulla peptide 8-22 (BAM8-22) andγ2-melanocyte stimulating hormone (γ2-MSH) activate this receptor andtrigger Ga_(g) mediated signaling (Lembo et al., 2002; Solinski et al.,Pharmacol Rev 66:570-597, 2014; Tatemoto et al., Biochem Biophys ResCommun 349:1322-8, 2006). Existing literature suggests that in mousemodels, receptors in the Mrgpr family modulate nociception andpruriception in vivo (Guan et al., Proc Natl Acad Sci USA 107:15933-8,2010; Liu et al., Cell 139:1353-65, 2009; Solinski et al., 2014). Arecent report showed that in humans, BAM8-22 produces itching sensationsthrough a histamine-independent pathway (Sikand et al., J Neurosci31:7563-7, 2011). Despite these studies, there still remain manyunanswered questions regarding the precise role of MrgprX1 in mediatingsomatosensory signals. Analysis of the coding region of the MrgprX1 generevealed genetic variation among humans (NHLBI GO Exome SequencingProject), shown in FIG. 1A and Table 1.

Naturally occurring variants of GPCRs have proved helpful inunderstanding differences in susceptibility to disease. The presentdisclosure provides compositions and methods for detecting MrgprX1missense mutations and determining the extent to which these MrgprX1missense mutations alter the pharmacological response of MrgprX1, forexample to the endogenous ligand BAM8-22.

A series of novel agonists (FIG. 1B) were developed to enable moredetailed characterization of signaling differences among MrgprX1variants. Lipidated constructs were generated corresponding to the twoactive MrgprX1 MTLs. Guided by the MTL results, PEG8 and palmitic acidwere covalently attached to the C-terminus of BAM8-22 and the N terminusof γ2-MSH to generate corresponding SMALs (Table 2). Methods ofgenerating lapidated peptides are disclosed in US20160052982, thecontents of which are incorporated herein by reference.

TABLE 2 Chemical structure of synthesized lipidated peptides. MolecularWeights (Da) Peptide Structure Calculated^(a) Observed^(b) LipidatedBAM8-22^(c)

2875.0 2872.7 Lipidated γ2-MSH^(d)

2231.2 2230.9 ^(a)Calculated molecular weights were estimated usingGenScript (Piscataway, NJ). ^(b)Observed molecular weights weredetermined by MALDI-TOF MS (Bruker microflex LT) in a positivereflectron mode using α-cyano-4-hydroxy cinnamic acid as the matrix.^(c)Lipidated BAM8-22 is comprised of BAM8-22 and a GGK spacer coupledto a PEG8 linker domain and palmitic acid. Note that the linker-lipidmodification is on the amino side chain group (N^(ε)) of the C-terminallysine. ^(d)Lipidated γ2-MSH is comprised of γ2-MSH coupled to a PEG8linker domain and palmitic acid. Note that the linker-lipid modificationis on the N terminus of the peptide. BAM8-22 (VGRPEWWMDYQKRYG) (SEQ IDNO: 1); BAM8-22 with GGK Spacer (VGRPEWWMDYQKRYGGGK) (SEQ ID NO: 2);γ2-MSH (YVMGHFRWDRFG) (SEQ ID NO: 3)

Previous work has shown that peptide ligands may be anchored to the cellsurface using recombinant DNA technology. Such membrane-tethered ligands(MTLs) provide a complementary tool to explore GPCR function (Fortin etal., PLoS One 6:e24693, 2011; Fortin et al., Proc Natl Acad Sci USA106:8049-54, 2009; Harwood et al., Mol Pharmacol 83:814-21, 2013).Conversion of these recombinant ligands into synthetic membrane anchoredligands (SMALs), in which a peptide is covalently coupled to a flexiblelinker and a lipid moiety, yields potent, soluble ligands that anchor tothe cell surface and activate the corresponding GPCR (Doyle et al., JBiol Chem 289:13385-96, 2014; Fortin et al., 2011). Potential advantagesof such ligands include increased potency and prolonged stability (Zhangand Bulaj, Curr Med Chem 19:1602-18, 2012).

In the present disclosure, this developed panel of ligands is used tocharacterize a series of MrgprX1 missense mutations with an allelefrequency exceeding 0.1% (Table 1).

TABLE 1 Allele frequencies of MgprX1 missense variants. All data werecollected from the NHLBI GO ESP Exome Variant Server. Variant dbSNPReference ID EA Frequency AA Frequency I36V rs11024885 0.63% 10.17% A46Trs78179510 17.69% 19.24% R55L rs55954376 0.01% 3.42% R131S rs1114481171.19% 0.23% H133R rs140351170 0.33% 0.07% H137R rs143702818 0.01% 0.41%F273L rs138263314 2.44% 0.53% Abbreviations: EA, European American; AA,African American.

A schematic representation of MrgprX1 (FIG. 1A) highlights the locationof each variant residue. The wild type amino acids in positions wheresequence variations occur are indicated by the single letter code. Thepresent compositions and methods demonstrate that two mutations inMrgprX1, R131S and H133R, alter receptor mediated signaling, resultingin loss and gain of function respectively. These variants may modifysusceptibility to histamine-independent itch and/or nociception.

Initial analysis of naturally occurring MrgprX1 variants with theendogenous ligand BAM8-22 identified R131S as a potentialloss-of-function mutation (FIG. 2). To further investigateligand-mediated signaling of this variant as well as other receptormutants, MTL and SMAL analogs of BAM8-22 and γ2-MSH were generated. Inaddition to confirming the loss of function resulting from the R131Smutation, use of these recombinant and synthetic ligands revealed thatthe H133R substitution conferred a ligand-dependent gain of functionphenotype (FIGS. 4A-4B and 5A-5B). Defining how missense mutations inthis receptor alter pharmacological function is an important first steptowards understanding the potential role of natural variants in alteringsomatosensation and/or the response to drugs targeting MrgprX1 in vivo.

There are multiple mechanisms through which missense mutations mayaffect GPCR function. Some variants affect the active/inactive stateequilibrium and may in turn have systematic effects on ligand-mediatedsignaling (Beinborn et al., Mol Pharmacol 65:753-60, 2004; Kopin et al.,Proc Natl Acad Sci USA 100:5525-30, 2003; Samama et al., J Biol Chem268:4625-4636, 1993). Other mutations alter ligand interaction with thereceptor, either directly or indirectly through changes in receptortertiary structure. (Bond et al., Proc Natl Acad Sci USA 95:9608-13,1998; Fortin et al., Mol Pharmacol 78:837-45, 2010).

The present disclosure provides that the R131S mutation decreases bothligand-mediated and ligand-independent (basal) activity of MrgprX1.These properties place it in the former group of mutations. Notably,these differences in receptor activity levels cannot be explained bychanges in receptor expression (FIGS. 7A-7B). The location of residueR131 in the second intracellular loop, a domain that has beenestablished as important in G protein binding (Hu et al., Nat Chem Biol6:541-8, 2010), demonstrates that this mutation could be affecting theability of MrgprX1 to interact with G proteins and/or shift MrgprX1 fromthe active to the inactive state.

The H133R mutation does not affect basal activity and slightly increasesthe efficacy of a subset of ligands (i.e. tethered and lipidated γ2-MSHbut not tethered or lipidated BAM8-22). This demonstrates that H133R isnot a systematic modulator and therefore belongs to the latter group ofmutations (as described above). Like with R131S, these changes inligand-mediated receptor activity are not accompanied by changes inreceptor expression. Given its location in the second intracellularloop, H133R may represent a mutation that impacts the ligand-receptorinteraction indirectly (e.g. by slightly altering the orientation ofresidues that interact with the ligand).

The purported role of MrgprX1 in mediating pain and somatosensation, inparticular histamine-independent itch (Bader et al., Pharmacol Rev66:1080-1105, 2014; Sikand et al., 2011; Solinski et al., 2014),indicates that the unique signaling properties of the R131S and H133Rvariants may have important implications for the development and use oftherapeutics targeting this receptor. Missense variants have also provenimportant in understanding differences in somatosensation previously.For example, the N40D mutation in the human opioid receptor (hMOR) mayalter susceptibility to pain (Lötsch and Geisslinger, Trends Mol Med11:82-9, 2005) and pruritus (Tsai et al., Acta Anaesthesiol Scand54:1265-1269, 2010). Similarly, missense mutations in the sodium channelNa_(v)1.7 have been linked to pain-related disorders (Drenth and Waxman,J Clin Invest 117:3603-9, 2007; Fertleman et al., Neuron 52:767-74,2006) and altered pain perception (Reimann et al., Proc Natl Acad SciUSA 107:5148-53, 2010).

The possibility that MrgprX1 variants may be linked to a specificphenotype highlights the need for data collection that will allow formatching of the MrgprX1 genotype with sensitivity to MrgprX1-mediatedsomatosensation. This should be feasible particularly with the R131Svariant, which has an allele frequency of greater than 1%. Futurestudies may reveal that mutations such as R131S are linked to decreasednociception or pruritus. Extending beyond the coding region of the gene,variations in upstream regulatory sequences may also play a role inaltering susceptibility to histamine-independent itch by alteringMrgprX1 expression (Wray, Nat Rev Genet 8:206-16, 2007).

The compositions of the present disclosure provide powerful molecularprobes to explore pharmacological differences between receptor variants.As illustrated, such modified peptide ligands (MTLs and their lapidatedcounterparts) exhibit enhanced effective concentration and thus provideexperimental tools that facilitate the pharmacological characterizationof GPCRs. In addition, MTLs can be expressed as transgenic constructsenabling exploration of corresponding receptor function in vivo (Harwoodet al., J Exp Biol 217:4091-8, 2014). Complementing such recombinantconstructs, lipidated peptides provide additional tools which can beapplied in vivo to probe receptor function and validate potentialtherapeutic targets (Doyle et al., 2014). Notably, activity of tetheredγ2-MSH and tethered BAM8-22 are recapitulated with their lipidatedanalogs, providing further support that MTLs are useful in predictingthe pharmacological properties of corresponding lipidated peptides.

Taken together, the compositions and methods of the present disclosuredemonstrate how naturally occurring missense variants may markedly alterthe pharmacological properties of a GPCR. In addition, the compositionsand methods of the present disclosure exemplify how MTLs and SMALsprovide complementary tools to differentiate receptor variants that aresystematic modulators from mutations that preferentially affect a subsetof receptor agonists. As with a growing number of GPCRs (Rana et al.,Annu Rev Pharmacol Toxicol 41:593-624, 2001; Thompson et al., MethodsMol Biol 1175:153-87, 2014), MrgprX1 receptor variants display importantdifferences in both basal and ligand-induced signaling that maycontribute to somatosensory variability in the human population.

Administration of the Compositions

The therapeutically effective amount of a composition according to thisdisclosure can vary within wide limits and may be determined in a mannerknown in the art. For example, the composition can be dosed according tobody weight. Such dosage will be adjusted to the individual requirementsin each particular case including the specific compound(s) beingadministered, the route of administration, the condition being treated,as well as the patient being treated. In another embodiment, the drugcan be administered by fixed doses, e.g., dose not adjusted according tobody weight. In general, in the case of oral or parenteraladministration to adult humans, a daily dosage of from about 0.5 mg toabout 1000 mg should be appropriate, although the upper limit may beexceeded when indicated. The dosage can be from about 5 mg to about 500mg per day, e.g., about 5 mg to about 400 mg, about 5 mg to about 300mg, about 5 mg to about 200 mg. The daily dosage can be administered asa single dose or in divided doses, or for parenteral administration itmay be given as continuous infusion.

A therapeutically effective amount of a composition is that whichprovides an objectively identifiable improvement as noted by theclinician or other qualified observer.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration. Thecompositions described herein can be administered orally, nasally,transdermally, pulmonary, inhalationally, buccally, sublingually,intraperintoneally, subcutaneously, intramuscularly, intravenously,rectally, intrapleurally, intrathecally, or parenterally. In oneembodiment, the compound is administered orally. One skilled in the artwill recognize the advantages of certain routes of administration.

The dosage regimen utilizing the compositions described herein isselected in accordance with a variety of factors including species,ethnicity, age, weight, sex and medical condition of the patient; theseverity of the condition to be treated; the route of administration;the renal and hepatic function of the patient; and the particularcomposition employed. An ordinarily skilled physician or veterinariancan readily determine and prescribe the effective amount of the drugrequired to prevent, counter, or arrest the progress of the condition.

Techniques for formulation and administration of the disclosedcompositions of the disclosure can be found in Remington: the Scienceand Practice of Pharmacy, 19^(th) edition, Mack Publishing Co., Easton,Pa. (1995). In an embodiment, the compounds described herein, and thepharmaceutically acceptable salts thereof, are used in pharmaceuticalpreparations in combination with a pharmaceutically acceptable carrieror diluent. Suitable pharmaceutically acceptable carriers include inertsolid fillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

Methods of Treatment

The compositions described herein can be used to treat a variety ofconditions including neuropathic pain, ocular pain, ocular inflammation,and dry eye.

In one aspect, the present disclosure provides a method of treatingneuropathic pain with the compositions described herein. Neuropathicpain according to the present disclosure is a pain initiated or causedby a primary lesion or dysfunction in the nervous system. Neuropathicpain according to the present disclosure could be divided into“peripheral” (originating in the peripheral nervous system) and“central” (originating in the brain or spinal cord). For example,neuropathic pain syndromes include postherpetic neuralgia (caused byHerpes Zoster), root avulsions, painful traumatic mononeuropathy,painful polyneuropathy (particularly due to diabetes), central painsyndromes (potentially caused by virtually any lesion at any level ofthe nervous system), postsurgical pain syndromes (e.g., postmastectomysyndrome, postthoracotomy syndrome, or phantom pain), and complexregional pain syndrome (e.g., reflex sympathetic dystrophy orcausalgia).

Neuropathic pain have typical symptoms like dysesthesias (spontaneous orevoked burning pain, often with a superimposed lancinating component),but the pain may also be deep and aching. Other sensations likehyperesthesia, hyperalgesia, allodynia (pain due to a normoxiousstimulus), and hyperpathia (particularly unpleasant, exaggerated painresponse) may also occur. The compositions of the present disclosure canbe administered to ameliorate at least one of these symptoms.

Current therapy for neuropathic pain aims only at reducing symptoms,generally by suppressing neuronal activity. Thus treatment options,e.g., non-steroidal anti-inflammatory drugs (NSAIDS), antidepressants,anticonvulsants, baclofen, neuromodulation modalities, or opiates,predominantly alleviate symptoms via nonspecific reduction of neuronalhyperexcitability rather than targeting the specific etiologies. Thecompositions of the present disclosure can be administered incombination with the current therapy for treating neuropathic pain. Forexample, the compositions of the present disclosure can be administeredin combination with an NSAID, an antidepressant, an anticonvulsant,baclofen, a neuromodulation modality, or an opiate for treatingneuropathic pain.

In another aspect, the present disclosure provides a method of treatingocular pain with the compositions described herein. Ocular pain can beco-incident with a number of conditions, including but not limited totrauma due to accidental or surgical injury, uveitis, dry eye, anddiabetic neuropathy. The standard of care for treatment of ocular painis typically either topically administered NSAIDs, or orallyadministered analgesic agents, such as NSAIDS or opioids likehydrocodone. In some embodiments, the compositions of the presentdisclosure can be administered in combination with an NSAID or opioidfor treating ocular pain.

In another aspect, the present disclosure provides a method of treatingocular inflammation with the compositions descried herein. Ocularinflammation can be caused by a microbial infection of the eye. Suchinfection may be fungal, viral or bacterial. Current therapies fortreating ocular inflammation include locally administered anti-cytokineor anti-inflammatory agents. In some embodiments, the compositions ofthe present disclosure can be administered in combination with ananti-cytokine or anti-inflammatory agent for treating ocularinflammation.

The compositions described herein can also be used to treat dry eye. Dryeye is primarily caused by the break-down of the pre-ocular tear filmwhich results in dehydration of the exposed outer surface. Withoutwishing to be bound by theory, there is a strong rationale that ocularinflammation as a result of pro-inflammatory cytokines and growthfactors plays a major role in the underlying causes of dry eye. As such,locally administered anti-cytokine or anti-inflammatory agents are oftenused in the treatment of dry eye. In some embodiments, the compositionsof the present disclosure can be administered in combination with ananti-cytokine or anti-inflammatory agent for treating dry eye.

With respect to combination therapies involving a first therapeuticagent (e.g., a composition of the present disclosure) and a secondtherapeutic agent (e.g., an anti-inflammatory agent, an opioid, anNSAID, or an antidepressant), the first therapeutic agent can beadministered concurrently with the second therapeutic agent; the firsttherapeutic agent can be administered before the second therapeuticagent; or the first therapeutic agent can be administered after thesecond therapeutic agent. The administrations of the first and secondtherapeutic agents can be separated by minutes or hours, e.g., one hour,two hours, three hours, four hours, five hours, or six hours.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. Although other methods andmaterials similar, or equivalent, to those described herein can be usedin the practice of the present invention, the preferred materials andmethods are described herein. It is to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

The terms “peptide,” “polypeptide,” and “protein” are usedinterchangeably herein and typically refer to a molecule comprising achain of two or more amino acids (e.g., most typically L-amino acids,but also including, e.g., D-amino acids, modified amino acids, aminoacid analogs, and amino acid mimetic). Peptides may be naturallyoccurring, synthetically produced, or recombinantly expressed. Peptidesmay also comprise additional groups modifying the amino acid chain, forexample, functional groups added via post-translational modification.Examples of post-translation modifications include, but are not limitedto, acetylation, alkylation (including methylation), biotinylation,glutamylation, glycylation, glycosylation, isoprenylation, lipoylation,phosphopantetheinylation, phosphorylation, selenation, and C-terminalamidation. The term peptide also includes peptides comprisingmodifications of the amino terminus and/or the carboxyl terminus.Modifications of the terminal amino group include, but are not limitedto, des-amino, N-lower alkyl, N-di-lower alkyl, and N-acylmodifications. Modifications of the terminal carboxy group include, butare not limited to, amide, lower alkyl amide, dialkyl amide, and loweralkyl ester modifications (e.g., wherein lower alkyl is C₁-C₄ alkyl).The term peptide also includes modifications, such as but not limited tothose described above, of amino acids falling between the amino andcarboxy termini. The term peptide can also include peptides modified toinclude one or more detectable labels.

The phrase “amino acid residue” as used herein refers to an amino acidthat is incorporated into a peptide by an amide bond or an amide bondmimetic.

The terminal amino acid at one end of the peptide chain typically has afree amino group (i.e., the amino terminus). The terminal amino acid atthe other end of the chain typically has a free carboxyl group (i.e.,the carboxy terminus). Typically, the amino acids making up a peptideare numbered in order, starting at the amino terminus and increasing inthe direction of the carboxy terminus of the peptide.

As used herein, the terms “treat,” “treating,” “treatment,” and the likerefer to reducing or ameliorating a disorder and/or a symptom associatedtherewith. It will be appreciated that, although not precluded, treatinga disorder or condition does not require that the disorder or symptomassociated therewith be completely eliminated. The terms “treat,”“treating,” or “treatment,” do not include prevention.

As used herein, a “subject” can be any mammal, e.g., a human, anon-human primate, mouse, rat, dog, cat, cow, horse, pig, sheep, goat,camel. In a preferred embodiment, the subject is a human.

As used herein, a “subject in need thereof” is a subject havingneuropathic pain, ocular pain, ocular inflammation, or dry eye.

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a solvent” includes a combination of two or moresuch solvents, reference to “a peptide” includes one or more peptides,or mixtures of peptides, reference to “a drug” includes one or moredrugs, reference to “a device” includes one or more devices, and thelike. Unless specifically stated or obvious from context, as usedherein, the term “or” is understood to be inclusive and covers both “or”and “and”.

Throughout the specification the word “comprising,” or variations suchas “comprises” or “comprising,” will be understood to imply theinclusion of a stated element, integer or step, or group of elements,integers or steps, but not the exclusion of any other element, integeror step, or group of elements, integers or steps.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can bealso be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clearfrom the context, all numerical values provided herein are modified bythe term “about.”

EXAMPLES Example 1

Materials and Methods

Generation of Receptor cDNA Constructs

The MrgprX1 cDNA, in pcDNA 3.1, was generously provided by Dr. XinzhongDong (Johns Hopkins University School of Medicine, Baltimore, Md.). Theconstruct was subcloned into pcDNA1.1 (Invitrogen). Naturally occurringmissense mutations were chosen using data from the NHLBI GO ESP ExomeVariant Server [Exome Variant Server, NHLBI Exome Sequencing Project(ESP), Seattle, Wash. (world wide web address atevs.gs.washington.edu/EVS/)]. Oligonucleotide-directed site-specificmutagenesis (Doyle et al., J Lipid Res 54:823-30, 2013; Fortin et al.,Proc Natl Acad Sci USA 106:8049-54, 2009) was used to generate thereceptor variants and corresponding epitope-tagged versions (where ahemagglutinin (HA) epitope tag was inserted immediately following theinitiator methionine). Forward and reverse DNA sequencing confirmed thecorrect nucleotide sequences for each construct.

Generation of Recombinant Membrane Tethered Ligands (MTLs)

A Membrane Tethered Ligand (MTL) is a cDNA-encoded protein consisting ofa peptide ligand fused to a transmembrane domain via a flexible linkerregion. Type I MTLs include a type I transmembrane domain, which orientsthe construct such that the N terminus of the ligand is extracellular.Conversely, type II MTLs result in an extracellular C terminus (Chou andElrod, Proteins 34:137-53, 1999; Harwood et al., Mol Pharmacol83:814-21, 2013). Corresponding DNA templates were used from previouslypublished tethered exendin (type I) and tethered chemerin (type II)constructs (Doyle et al., J Biol Chem 289:13385-96, 2014; Fortin et al.,2009). DNA sequences corresponding to the peptide ligands were eachsequentially replaced with those encoding BAM8-22 (VGRPEWWMDYQKRYG) (SEQID NO:1) and γ2-MSH (YVMGHFRWDRFG) (SEQ ID NO:3) (Lembo et al., 2002)using oligonucleotide-directed site-specific mutagenesis, producing bothtype I and type II MTLs for each peptide (Fortin et al., 2009; Harwoodet al., 2013).

Generation of Synthetic Membrane Anchored Ligands (SMAL) Constructs

Reagents for peptide synthesis were purchased from Chem-Impex (WoodDale, Ill.). N-Fmoc-PEG8-propionic acid, and palmitic acid were obtainedfrom AAPPTec (Louisville, Ky.) and Sigma-Aldrich (St. Louis, Mo.)respectively. Peptides were assembled on 4-hydroxymethylphenylacetamidomethyl (PAM) resin using the in situ neutralizationprotocol for N-Boc chemistry with2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU) as the activating agent on a 0.25 mmol scale (Schnölzer et al.,Int J Pept Res Ther 13:31-44, 2007). Peptide coupling reactions werecarried out with a 4-fold excess of the protected amino acid (1 mmol). AGGK peptide spacer was added to the C terminus of BAM8-22 to enablecoupling of the PEG8 linker.

After completion of the desired peptide sequence, coupling ofN-Fmoc-PEG8-propionic acid to the N terminus (γ2-MSH) or the C terminus(BAM8-22) preceded coupling of the lipid (palmitic acid) using standardactivation procedures (Doyle et al., 2014). Peptides were cleaved fromthe resin by using high HF conditions (90% anhydrous HF/10% anisole at0° C. for 1.5 h), and precipitated using cold Et₂O. Crude peptides werepurified by reversed phase HPLC, and the purities determined byanalytical HPLC [Vydac, C18, 5μ, 4 mm×250 mm] with a linear gradient ofsolvent B over 20 mins at a flow rate of 1 mL/min. Elution was monitoredby absorbance at 230 nm. Purities of peptides ranged from 90-95%.Peptides were analytically characterized by MALDI-TOF mass spectrometry.

Transfection and Luciferase Reporter Gene Assay

A luciferase reporter-based assay was utilized as an index ofreceptor-mediated signaling (as in Doyle et al., 2013). Human kidneycells (HEK293), grown in Dulbecco's modified Eagle's medium (DMEM)containing 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin wereseeded in 96-well plates and grown to 80% confluence. Usingpolyethylenimine (PEI, 2.0 μg/mL in serum-free DMEM), cells weretransiently transfected with cDNAs encoding (a) wild type or variantMrgprX1 (3 ng/well); (b) an SRE-luciferase PEST construct(SRE_(5x)-Luc-PEST), which includes five SRE repeats, a luciferasereporter gene, and the protein degradation sequence hPEST (Promega,catalog #E1340) (25 ng/well); and (c) a CMV-β-galactosidase construct asa control for variability in transfection efficiency (10 ng/well). Inexperiments that included transfection of an MTL-encoding construct, thecorresponding cDNA was added to the transfection mix, at 4 ng/well or asindicated.

Twenty-four hours following transfection, cells were stimulated withsoluble ligand for 4 hours (if applicable). After addition of SteadyLitereagent (PerkinElmer, Chicago, Ill.), luciferase activity of the lysatewas measured using a TopCount NTX plate reader. Subsequently,2-nitrophenyl β-D-galactopyranoside (ONPG) was added as a colorimetricsubstrate to enable quantification of β-galactosidase levels. Afterincubation with ONPG for 30 minutes, absorbance at 420 nm was measuredusing a SpectraMax microplate reader (Molecular Devices). Luciferaseactivity was normalized using the β-galactosidase activity data. Threeindependent experiments were performed, each with three technicalreplicates. Data were graphed and statistically analyzed using GraphpadPrism software.

Enzyme-Linked Immunosorbent Assay (ELISA)

An ELISA was used to assess total and surface receptor expression (as inDoyle et al., 2013). In brief, HEK293 cells were grown and seeded asabove using 96-well plates pretreated with poly-L-lysine. When 80%confluent, cells were transfected with HA-tagged receptor constructs.After 24 hours, the cells were fixed with 4% paraformaldehyde inphosphate buffered saline (PBS) for 10 min. To measure total expressionlevels, 0.1% Triton X-100 in PBS was applied in order to permeabilizethe cell membrane. To assess surface expression, treatment with TritonX-100 was omitted. Cells were washed with PBS/100 mM glycine and thenincubated in PBS/20% FBS for 30 minutes in order to block nonspecificantibody binding. A horseradish peroxidase (HRP)-conjugated antibodydirected against the HA epitope tag (Roche, catalog #12013819001) wasdiluted 1:500 and added to the cells for 3 hours. Cells were then washed5 times with PBS. The HRP substrate BM-blue(3,3′-5,5′-tetramethylbenzidine, Roche) was added at 50 μl per well.After 30 minutes, 50 μl of 2.0 M sulfuric acid was added to each well tostop the reaction. The concentration of the colorimetric product wasquantified by measuring absorbance at 450 nm using a SpectraMaxmicroplate reader (Molecular Devices).

Data Analysis

GraphPad Prism software version 6.0 (GraphPad Software Inc., La Jolla,Calif.) was used for sigmoidal curve fitting of ligandconcentration-response curves, linear regression, and statisticalanalysis. EC₅₀ and pEC₅₀ values were calculated for each independentexperiment as an index of ligand potency. Reported values represent themean of three independent experiments. Statistical comparisons were madeby one-way analysis of variance with Dunnett's multiple comparisonstest.

Example 2

Missense mutations in MrgprX1 result in differing levels of endogenouspeptide-mediated signaling.

Signaling of MrgprX1 following stimulation with the endogenous peptideligand BAM8-22 was measured using a luciferase reporter assay asdescribed herein. Cells expressing MrpgrX1 (WT or variant receptors)were stimulated for 4 hours with soluble BAM8-22. The R131S MrgprX1variant shows reduced endogenous ligand mediated signaling (FIG. 2).HEK293 cells were transfected with cDNA encoding either wild type orvariant MrgprX1, an SRE-luciferase reporter construct, andβ-galactosidase. After 24 hours, cells were stimulated with solubleBAM8-22 for 4 hours. Luciferase activity was quantified and normalizedrelative to β-galactosidase expression. Three independent experimentswere performed in triplicate, and data were expressed relative to thewild type receptor signal (maximum stimulation=100%). Results are shownas the mean±SEM. ****, p<0.0001 vs. WT (at 10⁻⁵ M).Concentration-response curves presented in FIG. 2 illustrate that six ofthe seven MrgprX1 variants assayed have a normal response to the ligand.However, the R131S variant exhibited lower levels of BAM8-22 mediatedactivity. The R131S best-fit curve is shifted to the right, suggesting asignificant loss of potency. Since soluble BAM8-22 does not fullystimulate the receptors when applied at the highest tested concentration(10 μM), accurate EC₅₀ values could not be calculated. It should benoted that HEK293 cells transfected with an empty vector control show noactivity after treatment with ligand (data not shown).

Example 3

Characterization of Novel Recombinant and Synthetic MrgprX1 Ligands.

As additional tools for structure-function studies, MTLs incorporatingone of two endogenous peptide ligands for MrgprX1, BAM8-22 and γ2-MSH,were generated. The activities of MTL constructs in both orientations(type I, with an extracellular N terminus of the ligand; type II, withan extracellular C terminus) were assessed using a luciferase-basedreporter assay as described herein. FIGS. 3A-3D show that Type Itethered BAM8-22 (FIG. 3A) and type II tethered γ2-MSH (FIG. 3B) areactive on the WT receptor. Lipidated BAM8-22 and lipidated γ2-MSHexhibit increased potency compared to the corresponding soluble peptides(FIG. 3C and FIG. 3D, respectively). To determine MTL activity, HEK293cells were transfected with increasing amounts of cDNA encoding eithertethered BAM8-22 or tethered γ2-MSH, as well as a fixed amount of cDNAencoding wild type MrgprX1, an SRE-luciferase reporter construct, andβ-galactosidase. To determine synthetic membrane anchored ligandactivity, similar methodology was utilized with tether cDNA omitted.Twenty-four hours after transfection, the cells were stimulated withlipidated BAM8-22 or lipidated γ2-MSH for 4 hours. Luciferase activitywas quantified and normalized relative to β-galactosidase expression.Data shown represent at least two independent experiments performed intriplicate. Results were expressed relative to the wild type receptorsignal (maximum=100%) and graphed as mean±SEM.

When expressed in HEK293 cells together with MrgprX1, a subset of MTLconstructs activated the receptor in a cDNA concentration-dependentmanner (FIG. 3A, 3B). Active MTLs included type I tethered BAM8-22 (freeextracellular N terminus) and type II tethered γ2-MSH (freeextracellular C terminus). These constructs were therefore used insubsequent experiments.

Using synthetic membrane anchored ligands (SMALs) which integrate intothe cellular membrane via a lipid moiety (Doyle et al., 2014; Fortin etal., PLoS One 6:e24693, 2011), recapitulating the activity ofrecombinant MTLs was attempted. Lipidated constructs were generatedcorresponding to the two active MrgprX1 MTLs. Guided by the MTL results,PEG8 and palmitic acid were covalently attached to the C-terminus ofBAM8-22 and the N terminus of γ2-MSH to generate corresponding SMALs(Table 2). When compared to the endogenous soluble form, both lipidatedBAM8-22 and lipidated γ2-MSH displayed significantly increased potency(FIGS. 3C, 3D).

In a parallel set of experiments (data not shown), signaling levels atsaturating concentrations of the four novel MrgprX1 ligands wereassessed at the WT receptor. Tethered BAM8-22, tethered γ2-MSH, andlipidated γ2-MSH signaling represented 38.4±5.4, 12.9±2.0, and 67.9%±2.4(mean±SEM) of maximum lipidated BAM8-22 signaling (at 10⁻⁷M),respectively.

Example 4

Select MrgprX1 missense mutations result in altered ligand mediatedsignaling.

The activity of tethered and lipidated BAM8-22 at each of the sevenMrgprX1 variants was assessed (FIGS. 4A-4B). The R131S variant displaysnegligible signaling levels with tethered BAM8-22 (FIG. 4A) as well asreduced signaling with lipidated BAM8-22 (FIG. 4B). To measure MTLactivity, HEK293 cells were transfected with cDNAs encoding tetheredBAM8-22, either wild type or variant MrgprX1, an SRE-luciferase reporterconstruct, and β-galactosidase. The empty vector, pcDNA1.1, wastransfected instead of receptor cDNA as a control. To measure syntheticmembrane anchored ligand activity, a similar methodology was utilizedwith the tether cDNA omitted. Cells were stimulated 24 hours aftertransfection with lipidated BAM8-22 for four hours. Luciferase activitywas quantified and normalized relative to 3-galactosidase expression.For each receptor, three independent experiments were performed intriplicate. Data are expressed relative to the maximum signal achievedat the wild type receptor. Results are shown as the mean±SEM. ****,p<0.0001 variant receptor vs. WT (at 10⁻⁶M in FIG. 4B). All variantsexcept R131S are significantly different from pcDNA1.1 (p<0.05).

Following stimulation with either the recombinant or the syntheticBAM8-22 analog, the R131S variant consistently displays attenuatedlevels of signaling. In addition to decreased efficacy, a statisticalanalysis of calculated EC₅₀ values for all seven variants suggests thatonly the R131S mutation significantly decreases the potency and efficacyof lipidated BAM8-22 (Table 3).

TABLE 3 Comparison of signaling induced by lipidated BAM8-22 at selectedMrgprX1 variants. Variant EC₅₀ (nM) pEC₅₀ ^(a) Curve Maximum^(a,b) WT12.0 7.92 ± 0.065 101.3% ± 2.7 I36V 9.3 8.03 ± 0.12 108.9% ± 5.4 A46T9.5 8.02 ± 0.112 111.6% ± 5.2 R55L 10.2 7.99 ± 0.142 107.5% ± 6.4 R131S57.1 7.24 ± 0.117****  40.4% ± 2.5**** H133R 8.6 8.06 ± 0.103 110.2% ±4.6 H137R 7.9 8.10 ± 0.089 109.2% ± 4.0 F273L 12.1 7.92 ± 0.076  95.9% ±3.1 ^(a)shown as mean ± SEM ^(b)curve maxima are extrapolated from thebest-fit curve. Luciferase signal at the WT receptor achieved at 10⁻⁶Mlipidated BAM8-22 is defined as 100%. ****p < 0.0001 (vs. WT)

The R131S variant also displays decreased ligand-mediated signaling witheither tethered or lipidated γ2-MSH (FIGS. 5A-5B). When stimulated withtethered (FIG. 5A) or lipidated (FIG. 5B) γ2-MSH, the R131S and H133Rvariants exhibit decreased and increased signaling levels compared tothe wild type receptor, respectively. To measure MTL activity, HEK293cells were transfected with cDNAs encoding tethered γ2-MSH, either wildtype or variant MrgprX1, an SRE-luciferase reporter construct, andβ-galactosidase. The empty vector, pcDNA1.1, was transfected instead ofreceptor cDNA as a control for background signaling. To measuresynthetic membrane anchored ligand activity, the tether cDNA wasomitted. Cells were stimulated 24 hours after transfection withlipidated γ2-MSH for four hours. Luciferase activity was quantified andnormalized relative to β-galactosidase expression. For each receptor,three independent experiments were performed in triplicate. Data areexpressed relative to the maximum signal achieved at the wild typereceptor. Results are shown as the mean±SEM. *, p<0.05; **, p<0.01;****, p<0.0001 vs. WT (at 10⁻⁷M in FIG. 5B). All variants except R131Sare significantly different from pcDNA1.1 (p<0.05). Additionally, theH133R mutation significantly increases tethered and lipidated γ2-MSHmediated signaling, an effect not observed with lipidated or tetheredBAM8-22. A moderate decrease in signaling with the R55L and F273Lvariants was observed with both tethered BAM8-22 and tethered γ2-MSH,although this decrease only reached statistical significance withtethered γ2-MSH.

Example 5

The R131S Missense Mutation Reduces the Basal Activity of MrgprX1.

To explore whether changes in receptor-mediated signaling levels in partreflect altered basal activity, ligand-independent signaling of theR131S and the H133R variants was assessed (FIG. 6). HEK293 cells weretransfected with cDNAs encoding the corresponding MrpgrX1 variant, anSRE-luciferase reporter construct, and β-galactosidase. After 24 hours,luciferase activity was quantified and normalized relative toβ-galactosidase expression. Three independent experiments were performedin triplicate. Data were expressed relative to the maximum signal onwild type MrgprX1 at 3 ng of cDNA, achieved by stimulating with 10⁻⁵ Msoluble BAM8-22 for four hours. Results are shown as the mean±SEM andlines were fitted with linear regression. ***, p<0.001 (vs. WT, at 8 ngcDNA). Wild type MrgprX1 exhibits significant basal activityapproximating 6% of the maximum BAM8-22 stimulated level of signaling(at 10 μM). The H133R variant shows basal activity levels comparable toWT. In contrast, the R131S variant shows markedly attenuatedligand-independent activity.

Example 6

Expression Levels of the R131S and H133R Variants are Comparable to WildType.

The possibility that the observed differences in ligand-dependent andligand-independent signaling were the result of altered receptorexpression was explored. An enzyme-linked immunosorbent assay (ELISA)was used for this analysis. Epitope-tagged versions of WT MrgprX1, andof the R131S and H133R variants were generated. Each receptor wasexpressed in HEK293 cells. Both the R131S and H133R variants exhibitlevels of total and surface expression comparable to WT MrgprX1 (FIGS.7A-7B). HEK293 cells were transfected with increasing amounts of cDNAencoding the respective N-terminally HA epitope tagged MrgprX1 variant.After 24 hours, surface (FIG. 7A) and total (FIG. 7B) expression levelswere assessed by ELISA using non-permeabilized and permeabilized cells,respectively. Differences between expression levels of the WT receptorand the R131S and H133R variants are not statistically significant(p>0.05). After subtraction of background signal (no cDNA transfected),data were expressed relative to maximum wild type MrgprX1 expression inpermeabilized cells (total expression). Results are shown as themean±SEM and lines were fitted with linear regression. These datasuggest that observed differences in signaling are not attributable tochanges in receptor expression.

Example 7

Production of a Lipidated BAM8-22 Construct (StablePeptide-Linker-Lipid) Targeting Human MrgprX1

In FIG. 8, a luciferase reporter gene assay in HEK293 cells was used toassess tethered ligand induced receptor activity. Cells were transientlyco-transfected with cDNAs encoding: MrgprX1, a tethered ligand (asindicated), an SRE-luciferase reporter gene (to assess Gαq signaling),and β-galactosidase control gene (to correct for interwell variability).Luciferase activity was assessed after 24 hours. FIG. 8 demonstratesthat recombinant membrane tethered BAM8-22 activates MrgprX1.

Based on design of the active BAM MTL (free extracellular N terminus ofthe peptide, anchored C terminus), a lipidated BAM construct wasgenerated. This lipidated analog (peptide-linker-lipid) comprises anendogenous BAM8-22 peptide, a PEG-8 linker with a KGG spacer, and apalmitic acid membrane anchor. In FIG. 9, a luciferase reporter geneassay in HEK293 cells was used to assess ligand (soluble or lipidated)induced receptor activity. Cells were transiently co-transfected withcDNAs encoding: human MrgprX1 or mouse MrgprC11, an SRE-luciferasereporter gene (to assess Gαq signaling), and β-galactosidase controlgene (to correct for interwell variability). Luciferase activity wasassessed after 4 hours.

FIG. 9 shows that the lipidated BAM8-22 analog (1-BAM′) has ˜100 foldhigher potency than its endogenous BAM8-22 counterpart (s-BAM′).Notably, the lipidated ligands are active on both human and mousereceptor orthologs which supports the validity of using mouse as an invivo model

The generation of a lipidated BAM8-22 construct with enhanced stabilityfollows a stepwise progression.

(i) Identification of Residues that can Tolerate Substitution.

In FIG. 10, a luciferase reporter gene assay in HEK293 cells was used toassess tethered ligand induced receptor activity. To define residuesimportant for ligand-receptor interaction, an alanine scan was performedon the BAM8-22 MTL. This provided an initial index of residues thatcould be modified without a significant loss of activity. In the sevenpositions where conversion to alanine resulted in complete loss offunction, we also made a series of conservative substitutions based onthe categorization of amino acids into the following subgroups: small,nucleophilic, hydrophobic, aromatic, acidic, and basic. With thisapproach, activity was restored in three of the seven constructs(positions 14, 16, 17; data not shown). In total, 11 positions in theBAM8-22 peptide were thus considered candidates for modification asoutlined below (FIG. 10).

(ii) Utilization of Predictive Algorithms to Guide Generation ofProtease Resistant Peptides.

The BAM8-22 sequence was analyzed using the Protease SpecificityPrediction Server (PROSPER) to identify protease sites. PROSPERrecognizes aspartic, cysteine, metallo, and serine protease families. Inour initial analysis of endogenous BAM8-22, cleavage sites for serineproteases (position 13 and position 14) as well as cysteine proteases(position 17) were observed (FIG. 11). Analysis of active MTLs from thealanine scan, illustrated that the M15A substitution resulted in removalof the cleavage site at position 13. When combined with an additionalmodification based on the conservative substitution experiments (Y17W),this double substituted analog (M15A-Y17W) showed full activity with nopredicated protease activity at either position 13 (serine protease) orposition 17 (cysteine protease. To remove the residual serine proteaseactivity at position 14, additional modifications will be introduced inthis position (described below). In FIG. 11, underlined residuesindicate positions predicted to be specifically cleaved by proteasefamily members. A luciferase reporter gene assay in HEK293 cells wasused to assess tethered ligand induced receptor activity.

(iii) Generation of Candidate Protease Resistant MTLs

Amino acid substitutions at position W14 will be incorporated into theM15A-Y17W MTL construct using oligonucleotide-directed, site-specificmutagenesis. Residue substitutions that will eliminate the one remainingserine protease site include; A, R, D, H, V, and P. As an alternativecombination of amino acids conferring protease resistance to BAM8-22,substitutions at position W14 will also be introduced into a M15A-D16EMTL. Analysis of this variant is predicted to remove similar proteasecleavage sites and is likely to be as active as endogenous BAM8-22 basedon conservative substitution experiments (data not shown).

(iv) Assessment of MTL Activity (Gαq Mediated Signaling)

HEK293 cells will be co-transfected with cDNA encoding the MTL variantsas outlined above, an SRE luciferase construct and either the humanMrgprX1 or the mouse ortholog. Activity will be quantified using aluciferase reporter gene. Several optimized peptide ligands which arepredicted to be protease resistant are expected to be identified. TheBAM analogs corresponding to the four most active MTLs will then besynthesized as soluble peptides to confirm protease resistance.

(v) Experimental Confirmation of Protease Resistance with SolublePeptides

The activity of four newly generated soluble ligands will be assessedafter a 24-hour incubation in the presence of live cells (e.g. HEK293cells or a neuronal cell line). The incubation results in an extendedexposure to endogenous peptidases. This methodology is well-establishedand has been utilized to assess a short, stable chemerin analog. FIG. 12illustrates the feasibility of this approach and the sustained activityof a stable chemerin analog in comparison to the corresponding short,endogenous sequence. In FIG. 12, a luciferase reporter gene assay inHEK293 cells was used to assess ligand-induced receptor activity.Ligands were either added fresh (no pre-incubation) or pre-incubatedwith cells overnight (exposure to endogenous peptidases) and thentransferred onto transfected cells. Luciferase activity was assessedafter a 4 hour stimulation with indicated ligand.

(vi) Chemical Synthesis of Protease Resistant SMALs

Based on the studies outlined above, up to four BAM variant sequenceswill be incorporated into the following cassette: peptide-PEG8linker-palmitic acid anchor. In brief, the constructs will be assembledusing standard Fmoc chemistry on solid phase resins. An amide or acidC-terminal end can be obtained based on the identity of the resin. Abifunctional (amine and acid derivatized) PEG8 linker will be used thatis protected on the amino end. After coupling to the peptide chain, theamine will be unmasked and coupled to the anchor possessing a free acidterminus. The entire construct will be cleaved from the solid phaseusing TFA/TES/H2O/EDT that results in the simultaneous removal of theside chain protecting groups. Compounds will be purified usingreversed-phase HPLC using binary gradients of H₂O and acetonitrilecontaining 0.1% TFA.

(vii) Pharmacological Assessment of SMALs

Up to four SMALs (as outlined above) will be characterized using theseries of assays described below. Control ligands will include: SMALpeptide minus lipid (to assess the contribution of lipidation toactivity) and a scrambled BAM8-22 peptide (negative control). Gαqsignaling: Efficacy and potency of SMALs will be determined using aluciferase reporter gene assay (outlined above) in HEK293 cellsexpressing either the human MrgprX1 receptor or the mouse ortholog. Washresistant activity: To determine the extent of anchoring, the luciferaseactivity assay will be completed with and without serial washing afteraddition of the ligand. Resistance to washing (an index of lipidanchoring) will be measured in cells incubated with ligand for 15minutes, washed 3 times using ligand free media, and further incubatedfor 4 hours.

In acknowledgement that species differences exist within the Mrgprs thatcan alter receptor mediated function, optimized ligands will be testedon both mouse and human receptors. Constructs that do not showinterspecies differences will be preferentially used for in vivoexperiments. A protease resistant sequence will likely be found usingthe recombinant MTL approach (encoding endogenous amino acids). Tofurther complement these efforts, backbone cyclization and incorporationof unnatural amino acids into SMALs at tolerated positions will furtherenhance stability. Based on the activity of the lipidated BAM8-22construct, altering the anchor is not anticipated. However, if furtheroptimization is required, the option of introducing other lipids intothe SMAL, including: unsaturated fatty acids to increase membranefluidity (e.g., oleic acid), cholesterol to target different domains ofthe membrane (e.g., lipid rafts), or alternative hydrocarbon anchors(e.g., stearic acid) to alter hydrophobic mismatch is possible.

Example 8

In Vivo Assessment of Modified BAM8-22.

To assess the therapeutic effectiveness of the compounds of the presentdisclosure on neuropathic pain, the compounds of the present disclosurewill be utilized in a mouse model of chronic constriction injury(CCI)-induced neuropathic pain. Previously, intrathecally appliedBAM8-22 (as a soluble agonist) was shown to attenuate mechanicalallodynia which was blocked in Mrgpr cluster knockout mice. Using amouse model of CCI, we show that soluble BAM8-22 inhibits neuropathicpain in vivo. FIG. 13 shows this effect 30 minutes after intrathecalinjection of ligand. Using a similar model system (spinal nerve ligationparadigm in rats), the effect of intrathecal BAM8-22 is transient andreturns to baseline 90 minutes after administration. In FIG. 13, BAM8-22 (0.5 mM, 5 μL, intrathecal) attenuates mechanical painhypersensitivity induced by CCI of the sciatic nerve. Paw withdrawfrequency of the ipsilateral hind paw to low-force (0.07 g) wasincreased 14-18 days post injury. Pain was reduced 30 minutes followingintrathecal administration of BAM8-22.

Briefly, after intrathecal administration of drug, a series of von Freyfilaments will be applied to the plantar surface of the hind paw and pawwithdrawal frequency of the ipsilateral hind paw determined (thecontralateral hind paw will be used as a control). There are severalconsiderations. It is anticipated that these lipidated analogs will showenhanced efficacy and longevity versus the prototype lipidated BAM8-22or its soluble counterpart. Endogenous soluble BAM8-22 already shown toreduce neuropathic pain and will be tested as a positive control.Lipidated BAM8-22 and additional compounds will be assessed forcomparison. In order to determine the length of effect of compounds invivo, multiple time points (30 min, lhr, 3 hr, 6 hr, 24 hr, and 3 days)will be assessed following intrathecal injection of ligand.

What is claimed is:
 1. A composition comprising a lipidated bovineadrenal medulla peptide 8-22 (BAM8-22) peptide analog.
 2. A compositioncomprising a BAM8-22 peptide, a PEG-8 linker with a KGG spacer, and apalmitic acid membrane anchor.
 3. The composition of claim 1 or 2,wherein the BAM8-22 peptide comprises the amino acid sequence of SEQ IDNO:1.
 4. The composition of claim 1 or 2, wherein the BAM8-22 peptideand the KGG spacer comprises the amino acid sequence of SEQ ID NO:2. 5.The composition of claim 1 or 2, wherein the BAM8-22 peptide comprisesat least one amino acid modification.
 6. The composition of claim 5,wherein the at least one amino acid modification reduces or inhibitsprotease activity.
 7. The composition of claim 5 or 6, wherein the atleast one amino acid modification is at position 15 of SEQ ID NO:1. 8.The composition of claim 5 or 6, wherein the at least one amino acidmodification is at position 17 of SEQ ID NO:1.
 9. The composition ofclaim 7, wherein the modification at position 15 of SEQ ID NO:1 is a Mto A substitution (M15A).
 10. The composition of claim 8, wherein themodification at position 17 of SEQ ID NO:1 is a Y to W substitution(Y17W).
 11. The composition of any one of claims 1-10, wherein theBAM8-22 peptide comprises at least two amino acid modifications.
 12. Thecomposition of claim 11, wherein the at least two amino acidmodifications are at positions 15 and 17 of SEQ ID NO:1.
 13. Thecomposition of claim 12, wherein the modification at position 15 of SEQID NO:1 is a M to A substitution (M15A) and the modification at position17 of SEQ ID NO:1 is a Y to W substitution (Y17W).
 14. The compositionof claim 2, wherein the PEG-8 linker and the palmitic acid membraneanchor are coupled to the amino side chain group of the C-terminallysine of the BAM8-22 peptide.
 15. The composition of any one of claims1-14, wherein the BAM8-22 peptide or analog thereof is cyclized.
 16. Acomposition comprising a lipidated γ2-melanocyte stimulating hormone(γ2-MSH) peptide analog.
 17. A composition comprising a γ2-MSH peptide,a PEG-8 linker, and a palmitic acid membrane anchor.
 18. The compositionof claim 16 or 17, wherein the γ2-MSH peptide comprises the amino acidsequence of SEQ ID NO:3.
 19. The composition of claim 17, wherein thePEG-8 linker and the palmitic acid membrane anchor are coupled to theN-terminus of the γ2-MSH peptide.
 20. The composition of any one ofclaims 16-19, wherein the γ2-MSH peptide or analog thereof is cyclized.21. A pharmaceutical composition comprising a composition of any one ofclaims 1-20 and a pharmaceutically acceptable carrier.
 22. A method oftreating neuropathic pain in a subject in need thereof comprisingadministering a therapeutically effective amount of a composition of anyone of claims 1-21.
 23. A method of treating ocular pain in a subject inneed thereof comprising administering a therapeutically effective amountof a composition of any one of claims 1-21.
 24. A method of treatingocular inflammation in a subject in need thereof comprisingadministering a therapeutically effective amount of a composition of anyone of claims 1-21.
 25. A method of treating dry eye in a subject inneed thereof comprising administering a therapeutically effective amountof a composition of any one of claims 1-21.
 26. The method of any one ofclaims 22-25, wherein the subject is a mammal.
 27. The method of claim26, wherein the mammal is a human.